Project Summary: The focus of this proposal is to develop an electrical/non-optical next generation sequencing technology to sequence a haplotype resolved human genome for less than $1,000. Recent advances in Next-Gen sequencing, along with the development of robust analysis methods, have given researchers the ability to determine the role of sequence variations in human diseases. However, the vast majority of sequencing technologies produce results that are limited to finding polymorphisms, and the importance of haplotypes has been largely neglected. In order to truly understand the genetic makeup of a specific disease there is a definite need to develop methods to identify the specific chromosome of all polymorphisms. This is because haplotypes are more effective for identifying disease-causing loci. To accomplish this goal we have defined three specific aims. Aim 1. We will develop a paired-end rolony sequencing strategy. Rolonies are rolling circle amplicon colonies. Rolonies are a very promising replacement for emulsion PCR beads. Rolonies are attractive due to their simplicity and small size. However, paired-end rolony sequencing is currently not possible using sequencing-by-synthesis, and we strongly believe paired-end reads are essential to allow for complete haplotype resolved assembly of a human genome. Therefore, aim 1 is devoted to establishing the paired-end sequencing approach. Our paired-end rolony sequencing strategy will be developed and tested using reversible terminators that are conjugated to a cleavable fluorescent moiety. We will then apply and optimize the paired-end sequencing strategy in our ISFET electrical sequencing device using reversible terminators without fluorescent labels. Aim 2. We will develop a sequencing strategy that allows for assembly of the human genome while maintaining haplotype information. We have termed our approach haplotype resolved whole genome sequencing (hrWGS). We believe that for human genome sequence information to be most valuable, haplotypes must be identified. Haplotypes are important for identifying the association between genetic variations and disease. Furthermore, haplotype information is a key factor in the genetic analysis of populations. Currently, not a single next- generation sequencing technology is designed to deliver haplotype information. We propose to develop two strategies for haplotype resolved sequencing. Aim 3. We will develop a non-light based sequencing strategy for paired-end rolonies. The non-light based strategy will employ a high density ISFET array to detect the biochemical events that occur when a base is incorporated during sequencing-by-synthesis. Our non-light based sequencing strategy offers tremendous improvements over existing technologies. Namely, we will combine rolony sequencing with innovative ISFET designs to allow for 10B sensors per chip. Additionally, we will utilize our innovative paired-end sequencing strategy and novel library preparation methods to allow for haplotype resolution. In conclusion, our innovative technology provides significant advances beyond currently available approaches and will significantly advance the current state-of-the-art. We estimate that we could sequence a haplotype resolved human genome for much less than $1,000 using the proposed technology.